Abstract
Diaryldienone derivatives with accessible β-carbons show strong anti-neoplastic properties, related to their ability to make covalent adducts with free thiols by Michael addition, and low toxicity in vivo. Accumulation of poly-ubiquitylated proteins, activation of the unfolded protein response (UPR) and induction of cell death are universal hallmarks of their activities. These compounds have been characterized as inhibitors of isopeptidases, a family of cysteine-proteases, which de-conjugate ubiquitin and ubiquitin-like proteins from their targets. However, it is unclear whether they can also react with additional proteins. In this work, we utilized the biotin-conjugated diaryldienone-derivative named 2c, as a bait to purify novel cellular targets of these small molecules. Proteomic analyses have unveiled that, in addition to isopeptidases, these inhibitors can form stable covalent adducts with different intracellular proteins, thus potentially impacting on multiple functions of the cells, from cytoskeletal organization to metabolism. These widespread activities can explain the ability of diaryldienone derivatives to efficiently trigger different cell death pathways.
Highlights
During the past decades the identification of new small molecule therapeutics has provided some improvements for clinical treatments in patients with various tumors
In order to better characterize the mechanisms through which, diaryldienone-derivatives partially-selective isopeptidase inhibitors (P-SIIs) trigger cell death and to define their cellular targets, we generated a biotinylated probe of 2c (Fig. 1). 2c is a P-SII that we have recently synthetized and optimized for in vivo delivery[8]
MEC-1 chronic lymphocytic leukemia cells were treated for different times with the two compounds and the appearance of cell death was evaluated by cytofluorimetric analysis
Summary
During the past decades the identification of new small molecule therapeutics has provided some improvements for clinical treatments in patients with various tumors. We refer to them as partially-selective isopeptidase inhibitors (P-SIIs). In cells treated with these P-SIIs, accumulation of polyubiquitylated proteins is evident, in partial analogy to bortezomib treatment[2,14]. Bortezomid/Carfilzomib are inhibitors of the proteasomal catalytic chamber approved for the use in clinic[9]. For this reason P-SIIs are usually considered as alternative proteasome inhibitors. The response to oxidative stress and protein misfolding are more pronounced in 2c compared to bortezomib treated cells[14]
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